Dual specificity polypeptide molecule

ABSTRACT

The present invention relates to a bispecific polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain providing a binding region derived from a T cell receptor (TCR) being specific for a major histocompatibility complex (MHC)-associated peptide epitope, and a binding region derived from an antibody capable of recruiting human immune effector cells by specifically binding to a surface antigen of said cells, as well as methods of making the bispecific polypeptide molecule, and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Application No.62/658,318, filed Apr. 16, 2018, U.S. Provisional Application No.62/532,713, filed Jul. 14, 2017, German application no. 102017115966.5filed 14 Jul. 2017, German Application no. 102017119866.0, filed 30 Aug.2017 and German Application No. 102018108995.3, filed Apr. 16, 2018, thecontent of which is incorporated herein by reference in its entirety.

This application is related to PCT/EP2018/069151 and PCT/EP2018/069157filed Jul. 13, 2018.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-087004_ST25.txt” createdon 13 Jul. 2018, and 193,732 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to a bispecific polypeptide moleculecomprising a first polypeptide chain and a second polypeptide chainproviding a binding region derived from a T cell receptor (TCR) beingspecific for a major histocompatibility complex (MHC)-associated peptideepitope, and a binding region derived from an antibody capable ofrecruiting human immune effector cells by specifically binding to asurface antigen of said cells, as well as methods of making thebispecific polypeptide molecule, and uses thereof.

BACKGROUND OF THE INVENTION

With the development of molecular cloning technology and the deepunderstanding of antibody engineering, there are diverse bispecificantibody formats (“bispecifics”) from which to choose in order toachieve the optimal biological activity and clinical purpose. In cancertherapy, bispecific antibodies have been developed with the purpose ofredirecting the activity of immune effector cells to the site of tumorthrough a first binding domain specific for an epitope on tumor cellsand a second binding domain specific for an epitope on the immuneeffector cells. Bispecific antibodies for retargeting of immune effectorcells have been developed in different formats, including formatswithout fragment crystallizable (Fc) region and IgG-derived formats withsymmetric or asymmetric design. Besides retargeting effector cells tothe site of cancer, new applications were established for bispecificantibodies. Bispecifics that can inhibit two correlated signalingmolecules at the same time can be developed to overcome inherent oracquired resistance and to be more efficient angiogenesis inhibitors. Inaddition, bispecific antibodies can be employed as promisingimmune-stimulatory agents to treat various diseases like cancer.Bispecific antibodies can also be used to treat hemophilia A bymimicking the function of factor VIII. Bispecific antibodies also havebroad application prospects in bone disorders and infections anddiseases of the central nervous system (reviewed in Yang F. et al.Bispecific Antibodies as a Development Platform for New Concepts andTreatment Strategies. Int J Mol Sci. 2016 Dec. 28; 18(1)).

T cells express T cell receptor (TCR) complexes that are able to induceantigen-specific immune responses. Engagement of antigen peptide/majorhistocompatibility complex (MHC) Class I on the target cell with the TCRinduces the formation of an immune synapse and leads to signalingthrough CD3 co-receptors, which are components of the TCR signalingcomplex. This signaling cascade directs T cell-mediated killing of thecell expressing the antigen through the release and transfer ofgranzymes and perforin from the T cell to the target cell.

Historically, discovery and production of single-chain connectedvariable domains of antibodies (scFvs, described by Bird et al. 1988)served as major driver for the development of bispecific antibodies.This concept finally led to generation of BiTE-molecules and theirclinical validation as a potent drug for the treatment of leukemia(Baeuerle, P. A.; Reinhardt, C. Bispecific T-cell engaging antibodiesfor cancer therapy. Cancer Res. 2009, 69, 4941-4944). In cancer,bispecific antibodies that co-engage the CD3 epsilon subunit and asurface antigen on the tumor cell trigger T cell-mediated killing of thetumor cell while circumventing the need for the direct interaction ofthe TCR and MHC class I in complex with antigen. This expands therepertoire of T cells able to recognize the tumor and act as effectorcells (Baeuerle, P. A.; Reinhardt, C. Bispecific T-cell engagingantibodies for cancer therapy. Cancer Res. 2009, 69, 4941-4944).

Stieglmaier J., et al. (in: Utilizing the BiTE (bispecific T-cellengager) platform for immunotherapy of cancer. Expert Opin Biol Ther.2015; 15(8):1093-9) describe that various approaches of T-cell-basedcancer immunotherapy are currently under investigation, among these areBiTE® (bispecific T-cell engager) antibody constructs, which have aunique design and mechanism of action. They are constructed bygenetically linking onto a single polypeptide chain the minimal bindingdomains of monoclonal antibodies for tumor-associated surface antigensand for the T-cell receptor-associated molecule CD3. Concurrentengagement of the target cell antigen and CD3 leads to activation ofpolyclonal cytotoxic T-cells, resulting in target cell lysis.Blinatumomab, a BiTE® targeting CD19, is being investigated in a broadrange of B-cell malignancies and has recently been approved in the USAby the US FDA for Philadelphia chromosome-negative relapsed/refractoryB-acute lymphoblastic leukemia under the trade name BLINCYTO™. The BiTE®platform is one of the clinically most advanced T-cell immunotherapyoptions.

However the shortcomings of small bispecific molecules, like BiTEs®,have been discovered to be poor production yields, difficultpurification processes, aggregation propensity and also a very shortserum half-life. To overcome the inherent limitations of this class ofmolecules various bispecific formats based on human IgG were developedstarting with the concept of recombinant bispecific prototypeimmunoglobulin (Ig)-G-like antibodies as devised more than two decadesago, when Morrison and colleagues fused flexible linker peptides to theC termini of the heavy chains of IgG followed by single-chain variabledomains with different binding specificities (Coloma, M. J. andMorrison, S. L. (1997) Design and production of novel tetravalentbispecific antibodies. Nat. Biotechnol. 15, 159-163). The moleculescould be differentiated from ‘normal’ antibodies because they had dualfunctionalities. Technical hurdles initially hampered furtherdevelopment, causing bispecific antibodies (bsAbs) to remain a topic ofR&D primarily in the academic and biotech environment. However, rapidlyevolving technologies that enabled the engineering, production, anddevelopment of recombinant protein derivatives, combined with renewedinterest from the pharmaceutical industry, jump-started the bsAbresearch field. Today, many different bsAb formats suitable for thedevelopment of therapeutic proteins are available (for reviews, seeGramer, mAbs. 2013; 5(6):962-973, Weidle, Cancer Genomics Proteomics.2013 November-December; 10(6):239-50, Brinkmann, MAbs. 2017February/March; 9(2):182-212.). In summary, the inclusion ofFc-(fragment crystalizable) parts, consisting of CH2 and CH3 domains ledto increased productivity, simplified purification processes andenhanced stability. In addition the serum half-life of such IgG-baseddrugs was prolonged due to i) the increase in size and ii) theinteraction of the Fc-part with the human Fc-receptor FcRn.

Development of IgG-based bispecific formats was further fueled by theadvent and incorporation of engineered mutations to facilitate thehetero-dimerization of two differing CH3-domains thereby connecting twodifferent polypeptide chains. The basic concept was introduced byRidgway J B, et al. (in: ‘Knobs-into-holes’ engineering of antibody CH3domains for heavy chain heterodimerization. Protein Eng. 1996 July;9(7):617-21) who disclosed the ‘knobs-into-holes’ approach as a noveland effective design strategy for engineering antibody heavy chainhomodimers for heterodimerization. In this approach a ‘knob’ variant wasfirst obtained by replacement of a small amino acid with a larger one inthe CH3 domain of a CD4-IgG immunoadhesin: T366Y. The knob was designedto insert into a ‘hole’ in the CH3 domain of a humanized anti-CD3antibody created by judicious replacement of a large residue with asmaller one: Y407T. The anti-CD3/CD4-IgG hybrid represents up to 92% ofthe protein A purified protein pool following co-expression of these twodifferent heavy chains together with the anti-CD3 light chain. Incontrast, only up to 57% of the anti-CD3/CD4-IgG hybrid is recoveredfollowing co-expression in which heavy chains contained wild-type CH3domains. Thus knobs-into-holes engineering facilitates the constructionof an antibody/immunoadhesin hybrid and likely other Fc-containingbifunctional therapeutics including bispecific immunoadhesins andbispecific antibodies. Atwell et al, 1997, J Mol Biol (Stableheterodimers from remodeling the domain interface of a homodimer using aphage display library) discloses a knob-into-hole mutation (knob:T366W/hole: T366S+L368A+Y407V) in the CH3 domain of the Fc domain forimproved heterodimerization. This concept was further improved by theadditional introduction of cysteine-residues to form a stabilizingdisulfide-bond between the heterodimeric CH3-domains as described byMerchant et al. 1998, Nature Biotechnology (An Efficient Route to HumanBispecific IgG).

Further concepts to produce heterodimeric molecules were disclosed byMuda et al. 2011, PEDS (Therapeutic assessment of SEED: a new engineeredantibody platform designed to generate mono- and bispecific antibodies);Gunasekaran et al. 2010, J Biol Chem (Enhancing antibody Fc heterodimerformation through electrostatic steering effects: applications tobispecific molecules and monovalent IgG), Moore et al. 2011, MAbs (Anovel bispecific antibody format enables simultaneous bivalent andmonovalent co-engagement of distinct target antigens); Von Kreudensteinet al. 2013, MAbs (Improving biophysical properties of a bispecificantibody scaffold to aid developability: quality by molecular design.)These concepts are summarized and reviewed by Ha et al. 2016, FrontImmunol (Immunoglobulin Fc Heterodimer Platform Technology: From Designto Application in Therapeutic Antibodies and Proteins) and Liu et al.2017, Front Immunol (Fc Engineering for Developing TherapeuticBispecific Antibodies and Novel scaffolds).

With the inclusion of Fc-parts consisting of Hinges, CH2 and CH3domains, or parts thereof, into bispecific molecules the problem ofunspecific immobilization of these molecules, induced by Fc: Fc-gammareceptor (FcgR) interactions arose. FcgRs are composed of different cellsurface molecules (FcgRI, FcgRIIa, FcgRIIb, FcgRIII) binding withdiffering affinities to epitopes displayed by Fc-parts of IgG-molecules.As such an unspecific (i.e. not induced by either of the two bindingdomains of an bispecific molecule) immobilization is unfavorable due toi) influence on pharmacokinetics of a molecule and ii) off-targetactivation of immune effector cells various Fc-variants and mutations toablate FcgR-binding have been identified.

Morgan et al. 1995, Immunology (The N-terminal end of the CH2 domain ofchimeric human IgG1 anti-HLA-DR is necessary for C1q, FcγRI and FcγRIIIbinding) disclose the exchange of the residues 233-236 of human IgG1with the corresponding sequence derived from human IgG2 resulting inabolished FcgRI binding, abolished C1q binding and diminished FcgRIIIbinding.

EP1075496 discloses antibodies and other Fc-containing molecules withvariations in the Fc region (233P, 234V, 235A and no residue or G inposition 236 and 327G, 330S and 331S) wherein the recombinant antibodyis capable of binding the target molecule without triggering significantcomplement dependent lysis, or cell mediated destruction of the target.

Dual affinity retargeting (DART) molecules are used in order to achieve,for example, an optimal redirected T-cell killing of B-cell lymphoma.The original DART technology is described in Moore et al. (in:Application of dual affinity retargeting molecules to achieve optimalredirected T-cell killing of B-cell lymphoma, Blood. 2011 Apr. 28;117(17):4542-51). Comparison with a single-chain, bispecific antibodybearing identical CD19 and CD3 antibody Fv sequences revealed DARTmolecules to be more potent in directing B-cell lysis. Further evolutionof the DART technology was achieved by the DART-Fc-molecules asdescribed in Root et al, 2016 antibodies (Development of PF-06671008, aHighly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule withExtended Half-Life for the Treatment of Cancer). This molecule combinedthe high potency of the DARTs with, among other positivecharacteristics, the extended serum half-life of Fc-based molecules.

The αβTCR (TCR) recognizes antigenic peptides presented by MHC and isresponsible for the specificity of T cells. Both α and β chains of theTCR possess variable (V) and constant domains. The V domains areinvolved in binding antigenic peptide and the constant domains traversethrough the T cell membrane. From crystal structure analysis of TCRbound to peptide-MHC complex, complementarity determining regions (CDR)3 of both the V_(α) and V_(β) chains preferably interact with peptide,while CDRs 1 and 2 interact with MHC. However, recognition of peptide byCDR 1 and recognition of MHC by CDR 3 has also been described(Piepenbrink et al, The basis for limited specificity and MHCrestriction in a T cell receptor interface, Nat Commun, 2013; 4, 1948).The TCR αβ heterodimer is closely associated with CD3 proteins, CD4 orCD8, and other adhesion and signal transducing proteins. Binding ofantigenic peptide by the TCR V regions triggers T cell activation bysignal transduction through the TCR constant domains via CD3 and CD4 orCD8 cytoplasmic proteins.

Single-chain TCRs (scTCRs) afford significant advantages in contrast tothe full-length TCR format for engineering, soluble protein expression,and clinical potential. From the perspective of soluble proteinexpression (i.e. manufacturing), scTCRs are produced as a singlepolypeptide, avoiding the requirement for production of each TCR chainas separate polypeptides and allowing for production of largerquantities of the properly assembled scTCR that binds to its peptide-MHCligand. This feature can allow for production yields that are necessaryfor clinical use. Finally, from the clinical perspective, scTCRsconsisting of only the V regions (scTv) can be formatted as therapeuticsor diagnostic reagents similar to scFv fragments.

US 2006-0166875 discloses a single chain T cell receptor (scTCR)comprising a segment constituted by a TCR alpha chain variable regionsequence fused to the N terminus of a TCR alpha chain constant regionextracellular sequence, a beta segment constituted by a TCR beta chainvariable region fused to the N terminus of a TCR beta chain constantregion extracellular sequence, and a linker sequence linking the Cterminus of the a segment to the N terminus of the beta segment, or viceversa, the constant region extracellular sequences of the alpha and betasegments being linked by a disulfide bond, the length of the linkersequence and the position of the disulfide bond being such that thevariable region sequences of the alpha and beta segments are mutuallyorientated substantially as in native alpha/beta T cell receptors.Complexes of two or more such scTCRs, and use of the scTCRs in therapyand in various screening applications are also disclosed. In contrast tothe scTCR described in US 2006-0166875, US 2012-0252742 discloses asoluble human single chain TCR without constant domains, consisting ofonly the variable fragments of the TCR (scTv), which is useful for manypurposes, including the treatment of cancer, viral diseases andautoimmune diseases.

McCormack E, et al (in: Bi-specific TCR-anti CD3 redirected T-celltargeting of NY-ESO-1- and LAGE-1-positive tumors. Cancer ImmunolImmunother. 2013 April; 62(4):773-85) disclose that NY-ESO-1 and LAGE-1are cancer testis antigens with an ideal profile for tumorimmunotherapy, combining up-regulation in many cancer types with highlyrestricted expression in normal tissues and sharing a common HLA-A*0201epitope, 157-165. They present data to describe the specificity andanti-tumor activity of a bifunctional ImmTAC, comprising a soluble,high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fusedto an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2,antigen-positive tumor cell lines, and freshly isolated HLA-A2- andLAGE-1-positive NSCLC cells. Employing in vivo optical imaging, theresults show in vivo targeting of fluorescently labelled high-affinityNYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors inxenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumormodel in which human lymphocytes were stably co-engrafted intoimmunodeficient NSG mice harboring tumor xenografts; efficacy wasobserved in both tumor prevention and established tumor models using aGFP fluorescence readout. Quantitative RT-PCR was used to analyze theexpression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall,LAGE-1 RNA was expressed at a greater frequency and at higher levelsthan NY-ESO-1 in the tumor samples. ImmTACs comprise a single-chain Fvderived from anti-CD3 antibody UCHT-1 covalently linked to the C- orN-terminus of the alpha or beta chain of the TCR.

EP1868650 is directed at diabody molecules and uses thereof in thetreatment of a variety of diseases and disorders, includingimmunological disorders, infectious disease, intoxication and cancers.The diabody molecules comprise two polypeptide chains that associate toform at least two epitope binding sites, which may recognize the same ordifferent epitopes on the same or differing antigens. Additionally, theantigens may be from the same or different molecules. The individualpolypeptide chains of the diabody molecule may be covalently boundthrough non-peptide bond covalent bonds, such as, but not limited to,disulfide bonding of cysteine residues located within each polypeptidechain. In particular embodiments, the diabody molecules further comprisean Fc region, which is disclosed herein as it allows engineering ofantibody-like properties (e.g. long half-life) into the molecule.EP1868650 requires the presence of binding regions of light chain orheavy chain variable domains of an immunoglobulin, and extensivelydiscusses functional Fc receptor binders.

WO 2016/184592 A1 discloses bispecific molecules in which onespecificity is contributed by a TCR and the other by an antibody, whichis directed against an antigen or epitope on the surface of lymphocytes,but does not disclose the specific arrangement of the elements of theTCR and the antibody variable regions as disclosed herein.

EP2258720A1 is directed to a functional T cell receptor (TCR) fusionprotein (TFP) recognizing and binding to at least one MHC-presentedepitope, and containing at least one amino acid sequence recognizing andbinding an antigen.

It is an object of the present invention to provide improved bispecificmolecules capable of targeting peptide-MHC-complexes, that can be easilyproduced, display high stability and also provide high potency whenbinding to the respective antigen epitopes. Other objects and advantagesof the present invention will become apparent when studying thefollowing description and the preferred embodiments thereof, as well asthe respective examples.

In a first aspect of the invention, the above object is solved byproviding a dual specificity polypeptide molecule selected from thegroup of molecules comprising a first polypeptide chain and a secondpolypeptide chain, wherein:

the first polypeptide chain comprises a first binding region of avariable domain (VD1) of an antibody specifically binding to a cellsurface antigen of a human immune effector cell, anda first binding region of a variable domain (VR1) of a TCR specificallybinding to an MHC-associated peptide epitope, anda first linker (LINK1) connecting said domains;the second polypeptide chain comprises a second binding region of avariable domain (VR2) of a TCR specifically binding to an MHC-associatedpeptide epitope, anda second binding region of a variable domain (VD2) of an antibodyspecifically binding to a cell surface antigen of a human immuneeffector cell, anda second linker (LINK2) connecting said domains;wherein said first binding region (VD1) and said second binding region(VD2) associate to form a first binding site (VD1)(VD2) that binds theepitope of the cell surface molecule;said first binding region (VR1) and said second binding region (VR2)associate to form a second binding site (VR1)(VR2) that binds saidMHC-associated peptide epitope;wherein said two polypeptide chains are fused to human IgG hinge domainsand/or human IgG Fc domains or dimerizing portions thereof; andwherein the said two polypeptide chains are connected by covalent and/ornon-covalent bonds between said hinge domains and/or Fc-domains; andwherein said dual specificity polypeptide molecule is capable ofsimultaneously binding the cell surface molecule and the MHC-associatedpeptide epitope, and dual specificity polypeptide molecules, wherein theorder of the binding regions in the polypeptide chains is selected fromVD1-VR1; VD1-VR2; VD2-VR1; VD2-VR2; VR1-VD1; VR1-VD2; VR2-VD1; VR2-VD2,and wherein the domains are either connected by LINK1 or LINK2.

Preferred is a dual specificity polypeptide molecule comprising a firstpolypeptide chain and a second polypeptide chain, wherein: the firstpolypeptide chain comprises a first binding region of a variable domain(VD1) derived from an antibody capable of recruiting human immuneeffector cells by specifically binding to a surface antigen of saidcells, and a first binding region of a variable domain (VR1) derivedfrom a TCR being specific for an MHC-associated peptide epitope, and afirst linker portion (LINK1) connecting the two domains; the secondpolypeptide chain comprises a second binding region of a variable domain(VR2) derived from a TCR being specific for an MHC-associated peptideepitope, and a second binding region of a variable domain (VD2) derivedfrom an antibody capable of recruiting human immune effector cells byspecifically binding to a surface antigen of said cells, and a secondlinker portion (LINK2) connecting the two domains; wherein said firstbinding region (VD1) and said second binding region (VD2) associate toform a first binding site (VD1)(VD2) that binds the epitope of the cellsurface molecule; said first binding region (VR1) and said secondbinding region (VR2) associate to form a second binding site (VR1)(VR2)that binds said MHC-associated peptide epitope; wherein at least one ofsaid polypeptide chains is connected at its c-terminus to hinge-regions,CH2 and/or CH3-domains or parts thereof derived from human IgG; andwherein said dual specificity polypeptide molecule is capable ofsimultaneously binding the immune effector cell antigen and theMHC-associated peptide epitope.

Preferably, the dual specificity polypeptide molecule according to thepresent invention binds with high specificity to both the immuneeffector cell antigen and a specific antigen epitope presented as apeptide-MHC complex, e.g. with a binding affinity (KD) of about 100 nMor less, about 30 nM or less, about 10 nM or less, about 3 nM or less,about 1 nM or less, e.g. measured by Bio-Layer Interferometry asdescribed in Example 6 or as determined by flow cytometry.

The inventive dual specificity polypeptide molecules according to thepresent invention are exemplified here by a dual specificity polypeptidemolecule comprising a first polypeptide chain comprising SEQ ID No. 16and a second polypeptide chain comprising SEQ ID No. 17.

In a second aspect of the invention, the above object is solved byproviding a nucleic acid(s) encoding for a first polypeptide chainand/or a second polypeptide chain as disclosed herein, or expressionvector(s) comprising such nucleic acid. In a third aspect of theinvention, the above object is solved by providing a host cellcomprising vector(s) as defined herein.

In a fourth aspect of the invention, the above object is solved byproviding a method for producing a dual specificity polypeptide moleculeaccording to the present invention, comprising suitable expression ofsaid expression vector(s) comprising the nucleic acid(s) as disclosed ina suitable host cell, and suitable purification of the molecule(s) fromthe cell and/or the medium thereof.

In a fifth aspect of the invention, the above object is solved byproviding a pharmaceutical composition comprising the dual specificitypolypeptide molecule according to the invention, the nucleic acid or theexpression vector(s) according to the invention, or the cell accordingto the invention, together with one or more pharmaceutically acceptablecarriers or excipients.

In a sixth aspect of the invention, the invention relates to the dualspecificity polypeptide molecule according to the invention, the nucleicacid(s) or the expression vector(s) according to the invention, the cellaccording to the invention, or the pharmaceutical composition accordingto the invention, for use in medicine.

In a seventh aspect of the invention, the invention relates to the dualspecificity polypeptide molecule according to the invention, the nucleicacid or the expression vector(s) according to the invention, the cellaccording to the invention, or the pharmaceutical composition accordingto the invention, for use in the treatment of a disease or disorder asdisclosed herein, in particular selected from cancer and infectiousdiseases.

In an eighth aspect of the invention, the invention relates to a methodfor the treatment of a disease or disorder comprising administering atherapeutically effective amount of the dual specificity polypeptidemolecule according to the invention, the nucleic acid or the expressionvector(s) according to the invention, the cell according to theinvention, or the pharmaceutical composition according to the invention.

In a ninth aspect of the invention, the invention relates to a method ofeliciting an immune response in a patient or subject comprisingadministering a therapeutically effective amount of the dual specificitypolypeptide molecule according to the invention or the pharmaceuticalcomposition according to the invention.

In a tenth aspect, the invention relates to a method of killing targetcells in a patient or subject comprising administering to the patient aneffective amount of the dual specificity polypeptide molecule accordingto the present invention.

As mentioned above, the invention provides new and improved dualspecificity polypeptide molecules. The molecules generally comprise afirst polypeptide chain and a second polypeptide chain, wherein thechains jointly provide a variable domain of an antibody specific for anepitope of an immune effector cell surface antigen, and a variabledomain of a TCR that is specific for an MHC-associated peptide epitope,e.g. cancer epitope or epitopes presented because of infection, e.g.viral infection, such as HIV. Antibody and TCR-derived variable domainsare stabilized by covalent and non-covalent bonds formed betweenFc-parts or portions thereof located on both polypeptide chains. Thedual specificity polypeptide molecule is then capable of simultaneouslybinding the cellular receptor and the MHC-associated peptide epitope.

In the context of the present invention, variable domains (VD1) and(VD2) are derived from antibodies capable of recruiting human immuneeffector cells by specifically binding to a surface antigen of saideffector cells. In one particular embodiment, said antibodiesspecifically bind to epitopes of the TCR-CD3 complex of human T cells,comprising the peptide chains TCRalpha, TCRbeta, CD3gamma, CD3delta,CD3epsilon, and CD3zeta.

The dual specificity polypeptide molecule according to the presentinvention comprise a first polypeptide and a second polypeptide chainproviding a first (VD1) and a second (VD2) binding region, respectively,of a variable domain derived from an antibody capable of recruitinghuman immune effector cells by specifically binding to a surface antigenof said cells. This first binding region (VD1) and said second bindingregion (VD2) associate to form a first binding site (VD1)(VD2) thatbinds the epitope of the immune effector cell surface antigen.Furthermore, the first and the second polypeptide chain of thepolypeptide molecule comprises a first (VR1) and a second (VR2) bindingregion, respectively, of a variable domain derived from a TCR beingspecific for an MHC-associated peptide epitope. Said first bindingregion (VR1) and said second binding region (VR2) associate to form asecond binding site (VR1)(VR2) that binds said MHC-associated peptideepitope. In one embodiment of the dual specificity polypeptide moleculeaccording to the invention, the order/orientation of the regions in thefirst polypeptide chain is selected from VD1-LINK1-VR1, andVR1-LINK1-VD1; in another embodiment, in the order/orientation of theregions in the second polypeptide chain is selected from VD2-LINK2-VR2,and VR2-LINK-VD2, that is, the arrangement of the binding sites can bere-arranged into a “left-handed” or “right-handed” molecule (see, forexample, FIG. 5). Furthermore, the configuration of the alpha and betachains of the TCR-related part can be switched.

In the context of the present invention, the dual affinity polypeptidemolecule according to the invention is exemplified by a construct thatbinds the SLYNTVATL peptide (SEQ ID No. 7) when presented as apeptide-MHC complex. Nevertheless, the concept of the invention isclearly not restricted to this particular peptide, and includesbasically any disease- or disorder related epitope that is presented inthe context with the MHC molecule. This presentation can be both MHCclass-I or -II related. Major histocompatibility complex class I (MHC-I)molecules are present on the surface of all nucleated cells and displaya large array of peptide epitopes for surveillance by the CD8⁺ T cellrepertoire. CD8⁺ T cell responses are essential for control andclearance of viral infections as well as for the elimination oftransformed and tumorigenic cells. Examples for preferred peptideepitopes to be recognized can be found in the respective literature, andespecially include the peptides as disclosed in tables 1 to 5 of WO2016/170139; tables 1 to 5 of WO 2016/102272; tables 1 or 2 of WO2016/156202; tables 1 to 4 of WO 2016/146751; table 2 of WO 2011/113819;tables 1 to 4b of WO 2016/156230; tables 1 to 4b of WO 2016/177784;tables 1 to 4 of WO 2016/202963; tables 1 and 2 of WO 2016/207164;tables 1 to 4 of WO 2017/001491; tables 1 to 4 of WO 2017/005733; tables1 to 8 of WO 2017/021527; tables 1 to 3 of WO 2017/036936; tables 1 to 4of PCT/EP2016/073416 for cancer treatment(s), U.S. Publication2016-0187351, U.S. Publication 2017-0165335, U.S. Publication2017-0035807, U.S. Publication 2016-0280759, U.S. Publication2016-0287687, U.S. Publication 2016-0346371, U.S. Publication2016-0368965, U.S. Publication 2017-0022251, U.S. Publication2017-0002055, U.S. Publication 2017-0029486, U.S. Publication2017-0037089, U.S. Publication 2017-0136108, U.S. Publication2017-0101473, U.S. Publication 2017-0096461, U.S. Publication2017-0165337, U.S. Publication 2017-0189505, U.S. Publication2017-0173132, U.S. Publication 2017-0296640, U.S. Publication2017-0253633, and U.S. Publication 2017-0260249, the contents of each ofthese applications are herein incorporated by reference in theirentireties. In another aspect, the dual affinity polypeptide moleculeaccording to the invention recognizes a peptide consisting of any ofthose peptides described in the aforementioned patent applications.

In an aspect, the dual affinity polypeptide molecule according to theinvention binds or is capable of specifically being recognized/bindingto one or more peptides with an overall length of from 8 to 100 aminoacids, from 8 and 30 amino acids, from 8 to 16 amino acids, preferablyfrom 8 and 14 amino acids, namely 8, 9, 10, 11, 12, 13, 14 amino acids,in case of the elongated class II binding peptides the length can alsobe 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. In yet another aspect,the dual affinity polypeptide molecule according to the invention bindsor is capable of specifically recognizing/binding to one more peptideswith an overall length of from 8 to 12 amino acids, from 8 to 10 aminoacids, from 9 to 15 amino acids, from 9 to 14 amino acids, from 9 to 13amino acids, from 9 to 12 amino acids, from 9 to 11 amino acids; from 10to 15 amino acids, from 10 to 14 amino acids, from 10 to 13 amino acids,or from 10 to 12 amino acids.

Other suitable epitopes can be identified from databases, such as, forexample, the Immune Epitope Database (available at www.iedb.org).

The term “human immune effector cell(s)” refers to a cell within thenatural repertoire of cells in the human immune system which, whenactivated, is able to bring about a change in the viability of a targetcell. The term “viability of a target cell” may refer within the scopeof the invention to the target cell's ability to survive, proliferateand/or interact with other cells. Such interaction may be either direct,for example when the target cell contacts another cell, or indirect, forexample when the target cell secretes substances which have an influenceon the functioning of another distant cell. The target cell may beeither native or foreign to humans. In the event that the cell is nativeto humans, the target cell is advantageously a cell which has undergonetransformation to become a malignant cell. The native cell mayadditionally be a pathologically modified native cell, for example anative cell infected with an organism such as a virus, a plasmodium or abacterium. In the event that the cell is foreign to humans, the targetcell is advantageously an invading pathogen, for example an invadingbacterium or plasmodium.

Preferred is the dual specificity polypeptide molecule according to theinvention, wherein said first and second polypeptide chains furthercomprise at least one hinge domain and/or an Fc domain or portionthereof. In antibodies, the “hinge” or “hinge region” or “hinge domain”refers to the flexible portion of a heavy chain located between the CH1domain and the CH2 domain. It is approximately 25 amino acids long, andis divided into an “upper hinge,” a “middle hinge” or “core hinge,” anda “lower hinge.” A “hinge subdomain” refers to the upper hinge, middle(or core) hinge or the lower hinge. The amino acids sequences of thehinges of an IgG1, IgG2, IgG3 and IgG4 molecule are (EU numberingindicated):

IgG1: (SEQ ID No. 1) E₂₁₆PKSCDKTHTCPPCPAPELLG IgG2: (SEQ ID No. 2)E₂₁₆RKCCVECPPCPAPPVAGP IgG3: (SEQ ID No. 3)ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE₂₁₆PKSCDTPPPCPRC PAPELLG IgG4:(SEQ ID No. 4) E₂₁₆SKYGPPCPSCPAPEFLG

The core hinge region usually contains at least one cysteine-bridgeconnecting the two heavy chains. Furthermore, mutations can be made inthe lower hinge region to ameliorate unwanted antibody-dependentcell-mediated cytotoxicity (ADCC).

Preferred is a dual specificity polypeptide molecule according to thepresent invention, comprising at least one IgG fragment crystallizable(Fc) domain, i.e. a fragment crystallizable region (Fc region), the tailregion of an antibody that interacts with Fc receptors and some proteinsof the complement system. Fc regions contain two or three heavy chainconstant domains (CH domains 2, 3, and 4) in each polypeptide chain. TheFc regions of IgGs also bear a highly conserved N-glycosylation site.Glycosylation of the Fc fragment is essential for Fc receptor-mediatedactivity. The small size of bispecific antibody formats such as BiTEs®and DARTs (˜50 kD) can lead to fast clearance and a short half-life.Therefore, for improved pharmacokinetic properties, the scTv-cellularreceptor (e.g. CD3) dual specificity polypeptide molecule can be fusedto a (human IgG1) Fc domain, thereby increasing the molecular mass.Several mutations located at the interface between the CH2 and CH3domains, such as T250Q/M428L and M252Y/S254T/T256E+H433K/N434F, havebeen shown to increase the binding affinity to neonatal Fc receptor(FcRn) and the half-life of IgG1 in vivo. By this the serum half-life ofan Fc-containing molecule could be further extended.

In the dual specificity polypeptide molecules of the invention, said Fcdomain can comprises a CH2 domain comprising at least one effectorfunction silencing mutation. Preferably, these mutations are introducedinto the ELLGGP (SEQ ID No. 50) sequence of human IgG1 (residues233-238) or corresponding residues of other isotypes) known to berelevant for effector functions. In principle, one or more mutationscorresponding to residues derived from IgG2 and/or IgG4 are introducedinto IgG1 Fc. Preferred are: E233P, L234V, L235A and no residue or G inposition 236. Another mutation is P331S. EP1075496 discloses arecombinant antibody comprising a chimeric domain which is derived fromtwo or more human immunoglobulin heavy chain CH2 domains, which humanimmunoglobulins are selected from IgG1, IgG2 and IgG4, and wherein thechimeric domain is a human immunoglobulin heavy chain CH2 domain whichhas the following blocks of amino acids at the stated positions: 233P,234V, 235A and no residue or G in position 236 and 327G, 330S and 331Sin accordance with the EU numbering system, and is at least 98%identical to a CH2 sequence (residues 231-340) from human IgG1, IgG2 orIgG4 having said modified amino acids.

Examples of preferred CH2 partial sequences to be used can be (fully orpartially) as follows:

(SEQ ID No. 5) 231-AP PVA- GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCK VSNKALPA S IEK-334;and (SEQ ID No. 6) 231-AP PVA- GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPA S IEK-334,with the changes underlined, that in position 297 carry an N(glycosylated variant) or a residue selected from the group of A, G andQ (deglycosylated variant).

In the dual specificity polypeptide molecules of the invention, said Fcdomain can comprise a CH3 domain comprising at least one mutationfacilitating the formation of heterodimers. To maximize yield of thedesired heterodimeric dual specificity-Fc protein and to simplifypurification, “knobs-into-holes” mutations can be engineered into the Fcdomain. With this design, Fc domains are driven to form heterodimersinstead of their normal homodimers by addition of protruding bulkyhydrophobic residues (“knobs”) to one chain and creation ofcomplementary hydrophobic pockets (“holes”) on the other. A ‘knob’variant can be obtained by replacement of a small amino acid with alarger one to insert into a ‘hole’ in the opposite domain created byreplacement of a large residue with a smaller one (Ridgway, J. B. B.;Presta, L. G.; Carter, P. “Knobs-into-holes” engineering of antibody CH3domains for heavy chain heterodimerization. Protein Eng. 1996, 9,617-621; WO 2002/002781).

Preferred is a dual specificity polypeptide molecule according to theinvention, wherein said knob-into-hole mutation is selected from T366Was knob, and T366'S, L368′A, and Y407′V as hole in the CH3 domain (see,e.g. WO 98/50431). This set of mutations can be further extended byinclusion of the mutations K409A and F405′K as described by Wei et al.(Structural basis of a novel heterodimeric Fc for bispecific antibodyproduction, Oncotarget. 2017). Another knob can be T366Y and the hole isY407′T.

The dual specificity polypeptide molecules of the invention canfurthermore comprise artificially introduced cysteine bridges between atleast one cysteine residue on the first polypeptide chain and at leastone cysteine residue on the second polypeptide chain in order to improvethe stability of the molecules, optimally without interfering with thebinding characteristics of the bivalent molecule, and/or for improvedheterodimerization. For added stability, a disulfide bond can beintroduced through the addition of a single cysteine in the CH3 domainof both the knob and hole chains. Preferred is the dual specificitypolypeptide molecule according to the invention, wherein the Fc domaincomprises a CH3 domain comprising at least one additional cysteineresidue, for example S354C and/or Y349C.

Preferred is a dual specificity polypeptide molecule according to theinvention wherein said CD molecule is selected from the group of immuneresponse-related CD molecules, CD3, such as the CD3γ, CD3δ, and CD3εchains, CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25,CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49,CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134,CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46,NKG2D, GITR, FccRI, TCRalpha/beta, TCRgamma/delta and HLA-DR. Dependingon the combination of the two antigen binding entities of the dualspecificity polypeptide molecule according to the invention, specificadvantages regarding the function of the molecule, in particular anenhanced activity can be achieved.

Preferred is the exemplary dual specificity polypeptide moleculeaccording to the invention, wherein the regions in the first polypeptidechain SEQ ID No. 28 for VD1, SEQ ID No. 29 for VR1, SEQ ID No. 30 forLINK1; and the regions in the second polypeptide chain comprise SEQ IDNo. 31 for VD2, SEQ ID No. 32 for VR2, and SEQ ID No. 30 for LINK2.

Further preferred is the exemplary dual specificity polypeptide moleculeaccording to the invention, wherein the FC region in the firstpolypeptide chain comprises SEQ ID No. 26 (Fc1), and the FC region inthe second polypeptide chain comprises SEQ ID No. 27 (Fc2).

Further preferred is the exemplary dual specificity polypeptide moleculeaccording to the invention comprising a first polypeptide chaincomprising SEQ ID No. 16 (1. chain of full molecule) and a secondpolypeptide chain comprising SEQ ID No. 17 (2. chain of full molecule).

Even further preferred is the exemplary dual specificity polypeptidemolecule according to the invention, wherein said first binding site(VD1)(VD2) that binds the epitope of the surface antigen of human immunecells (e.g. CD3) is humanized; and/or said second binding site(VR1)(VR2) that binds said MHC-associated peptide epitope is affinitymaturated.

Humanized antibodies are antibodies (or parts thereof) from non-humanspecies whose protein sequences have been modified to increase theirsimilarity to antibody variants produced naturally in humans. Theprocess of “humanization” is usually applied to monoclonal antibodiesdeveloped for administration to humans (for example, antibodiesdeveloped as anti-cancer drugs). Suitable methods for humanization areknown from the literature, and, for example, reviewed in Olimpieri, PierPaolo, Paolo Marcatili, and Anna Tramontano. “Tabhu: Tools for AntibodyHumanization.” Bioinformatics 31.3 (2015): 434-435. PMC; Safdari Y,Farajnia S, Asgharzadeh M, Khalili M. Antibody humanization methods—areview and update. Biotechnol Genet Eng Rev. 2013; 29:175-86; orAhmadzadeh V, Farajnia S, Feizi M A, Nejad R A. Antibody humanizationmethods for development of therapeutic applications. Monoclon AntibImmunodiagn Immunother. 2014 April; 33(2):67-73.

In general, in vitro affinity maturation of TCRs and antibodies can bedone according to methods described in the literature, in particularusing yeast or phage surface display (based on, for example, Holler P D,et al. In vitro evolution of a T cell receptor with high affinity forpeptide/MHC. Proc Natl Acad Sci USA. 2000 May 9; 97(10):5387-92; Boder ET et al., Directed evolution of antibody fragments with monovalentfemtomolar antigen-binding affinity. Proc Natl Acad Sci USA. 2000 Sep.26; 97(20):10701-5; and, as a recent example, Zhao Q, et al. Affinitymaturation of T-cell receptor-like antibodies for Wilms tumor 1 peptidegreatly enhances therapeutic potential. Leukemia. 2015;29(11):2238-2247).

The binding sites (VD1)(VD2) and (VR1)(VR2) of the present descriptionpreferably specifically bind to a surface antigen of human immune cellsand a peptide-HLA molecule complex, respectively. As used herein inconnection with binding sites of the present description, “specificbinding” and grammatical variants thereof are used to mean a site havinga binding affinity (KD) for a peptide-HLA molecule complex and/or anantibody epitope of 100 μM or less.

The binding sites (VD1)(VD2) and (VR1)(VR2) of the present descriptionbind to a CD antibody epitope or a peptide-HLA molecule complex,respectively, with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity binding sites having binding affinities ofabout 1 μM or less, about 100 nM or less, about 50 nM or less, about 25nM or less, about 10 nM or less, about 5 nM or less, about 2 nM or less,about 1 nM or less, about 500 μM or less, about 200 μM or less, about100 μM or less Non-limiting examples of preferred binding affinityranges for binding sites of the present invention include about 10 μM toabout 100 μM, 100 μM to about 1 nM, 1 nM to about 10 nM, about 10 nM toabout 20 nM, about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM, e.g. measured by Bio-Layer Interferometryas described in Example 6.

In an aspect, the disclosure provides for a polypeptide having at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence described herein, for example, aminoacid sequences 1 to 58. In another aspect, the disclosure provides for afirst or second polypeptide having at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to an amino acidsequence described herein. In yet another aspect, the disclosureprovides for a duel specific polypeptide molecule having a sequenceidentity of at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to one or more amino acid sequencesdescribed herein. The disclosure further provides for aspects whereinthe percent identity of at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% applies to any of the sequences of thestructural regions described in FIG. 1, for example, VD1, VR1, Link1,VR2, VD2, Link2, or hinge region, and as described or being part of thesequences as disclosed herein.

In an aspect, polypeptides or duel specific polypeptide moleculesdescribed herein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions of one or moreamino acids. In another aspect, polypeptides or duel specificpolypeptide molecules described herein may include 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more amino acidsubstitutions, deletions or insertions. In yet another aspect,polypeptides or duel specific polypeptide molecules described herein mayinclude 1 to 5, 1 to 10, 1 to 20, 2 to 5, 2 to 10, 5 to 20, 5 to 50, or10 to 100 amino acid substitutions, deletions or insertions. In anaspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or50, or more amino acid substitutions, deletions or insertions applies toany of the structural regions described in FIG. 1, for example, VD1,VR1, Link1, VR2, VD2, Link2, or hinge regions. The disclosure furtherprovides for aspects wherein 1 to 5, 1 to 10, 1 to 20, 2 to 5, 2 to 10,5 to 20, 5 to 50, or 10 to 100 amino acid substitutions, deletions orinsertions applies to the sequences of any of the structural regionsdescribed in FIG. 1, for example, VD1, VR1, Link1, VR2, VD2, Link2, orhinge region, and as described or being part of the sequences asdisclosed herein.

In an aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,or 50 or more amino acids may be added to the N-terminus or C-Terminusof a polypeptide or duel specific polypeptide molecule described herein,for example, amino acid sequences 1 to 58.

In an aspect, VD1 may have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to the amino acid sequence of SEQ ID NO: 28.

In an aspect, VR1 may have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to the amino acid sequence of SEQ ID NO: 29.

In an aspect, LINK1 or LINK2 may have at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to the amino acid sequence of SEQ ID NO: 30.

In an aspect, VD2 may have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to the amino acid sequence of SEQ ID NO: 31.

In an aspect, VR2 may have at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to the amino acid sequence of SEQ ID NO: 32.

In an aspect, hinge may have at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, or SEQ ID NO: 4. In an aspect, CH2 domain may have at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% identity to the amino acid sequence ofSEQ ID NO: 5 or SEQ ID NO: 6.

In an aspect, Fc region may have at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO:27.

In an aspect, the disclosure provides for a polypeptide having at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 43, 44, 45, or 46.

In an aspect, the polypeptides or duel specific polypeptide molecules asdisclosed herein can be modified by the substitution of one or moreresidues at different, possibly selective, sites within the polypeptidechain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

In another preferred embodiment of the dual specificity polypeptidemolecule according to the invention, said molecule carries an activeagent or a portion thereof that is coupled or conjugated thereto. Saidactive agent can be selected from the group consisting of a detectablelabel, an immunostimulatory molecule, and a therapeutic agent.

The detectable label can be selected from the group consisting ofbiotin, streptavidin, an enzyme or catalytically active fragmentthereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or afluorescent, phosphorescent, or chemiluminescent molecule. Detectablelabels for diagnostic purposes include for instance, fluorescent labels,radiolabels, enzymes, nucleic acid probes and contrast reagents.

Therapeutic agents which may be associated with the molecules of theinvention include immunomodulators, radioactive compounds, enzymes(perforin for example), chemotherapeutic agents (cis-platin forexample), or a toxin. Other suitable therapeutic agents include smallmolecule cytotoxic agents, i.e. compounds with the ability to killmammalian cells having a molecular weight of less than 700 Daltons. Suchcompounds could also contain toxic metals capable of having a cytotoxiceffect. Furthermore, it is to be understood that these small moleculecytotoxic agents also include pro-drugs, i.e. compounds that decay orare converted under physiological conditions to release cytotoxicagents. Examples of such agents include cis-platin, maytansinederivatives, rachelmycin, calicheamicin, docetaxel, etoposide,gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimersodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate,auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e.proteins or fragments thereof with the ability to kill mammalian cells.For example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A,DNase and RNase; radio-nuclides, i.e. unstable isotopes of elementswhich decay with the concurrent emission of one or more of α or βparticles, or γ rays. For example, iodine-131, rhenium-186, indium-111,yttrium-90, bismuth-210 and -213, actinium-225 and astatine-213;chelating agents may be used to facilitate the association of theseradio-nuclides to the molecules, or multimers thereof;immuno-stimulants, i.e. immune effector molecules which stimulate immuneresponse. For example, cytokines such as IL-2 and IFN-γ, chemokines suchas IL-8, platelet factor 4, melanoma growth stimulatory protein,complement activators; or xenogeneic protein domains, allogeneic proteindomains, viral/bacterial protein domains, viral/bacterial peptides.

Another aspect of the present invention then relates to a nucleic acidmolecule encoding for a first polypeptide chain and/or a secondpolypeptide chain as disclosed herein, or an expression vectorcomprising such a nucleic acid. The nucleic acid molecule can be a DNA,cDNA, PNA, RNA, and combinations thereof. The nucleotide sequence codingfor a particular peptide, oligopeptide, or polypeptide may be naturallyoccurring or they may be synthetically constructed. Generally, DNAsegments encoding the peptides, polypeptides, and proteins of thisinvention are assembled from cDNA fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene that is capable of being expressed in a recombinant transcriptionalunit comprising regulatory elements derived from a microbial or viraloperon. The term “expression product” means the polypeptide or proteinthat is the natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s). The term “fragment”, whenreferring to a coding sequence, means a portion of DNA comprising lessthan the complete coding region, whose expression product retainsessentially the same biological function or activity as the expressionproduct of the complete coding region. Depending on the intended use,the nucleic acid can be codon-optimized for expression in a suitable(e.g. microbial) host cell. Redundancy in the genetic code allows someamino acids to be encoded by more than one codon, but certain codons areless “optimal” than others because of the relative availability ofmatching tRNAs as well as other factors (Gustafsson et al., 2004).

The nucleic acid may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and may or may notcontain introns so long as it codes for the polypeptide chains.

The nucleic acid (e.g. DNA) may then be comprised and/or expressed in asuitable host to produce a polypeptide comprising the polypeptide chainof the invention. Thus, the nucleic acid (e.g. DNA) encoding thepolypeptide chain of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention, as is known in the art. The nucleicacid (e.g. DNA, or in the case of retroviral vectors, RNA) encoding thepolypeptide chain(s) constituting the compound of the invention may bejoined to a wide variety of other nucleic acid (e.g. DNA) sequences forintroduction into an appropriate host. The companion nucleic acid willdepend upon the nature of the host, the manner of the introduction ofthe DNA into the host, and whether episomal maintenance or integrationis desired. Generally, the nucleic acid is inserted into an expressionvector, such as a plasmid, in proper orientation and correct readingframe for expression. If necessary, the nucleic acid may be linked tothe appropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host using standard techniques. Generally, notall of the hosts will be transformed by the vector. Therefore, it willbe necessary to select for transformed host cells. One selectiontechnique involves incorporating into the expression vector a nucleicacid sequence, with any necessary control elements, that codes for aselectable trait in the transformed cell, such as antibiotic resistance.Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell. Host cellsthat have been transformed by the recombinant nucleic acid of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

In one embodiment, the description provides a method of producing amolecule as described herein, the method comprising culturing a hostcell capable of expressing the polypeptide chain(s) under conditionssuitable to promote expression of said chain(s).

In one aspect, to obtain cells expressing molecules of the presentdescription, nucleic acids encoding polypeptide chains comprisingTCR-alpha and/or TCRbeta binding domains are cloned into expressionvectors, such as gamma retrovirus or lentivirus. In another aspect, toobtain cells expressing molecules of the present description, RNAs aresynthesized by techniques known in the art, e.g., in vitro transcriptionsystems. The in vitro-synthesized RNAs are then introduced into suitablecells by electroporation to express polypeptide chains.

To increase the expression, nucleic acids encoding chains of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed. In addition to strong promoters,expression cassettes of the present description may contain additionalelements that can enhance transgene expression, including a centralpolypurine tract (cPPT), which promotes the nuclear translocation oflentiviral constructs (Follenzi et al., 2000), and the woodchuckhepatitis virus posttranscriptional regulatory element (wPRE), whichincreases the level of transgene expression by increasing RNA stability(Zufferey et al., 1999).

The alpha and beta binding domain chains of a molecule of the presentinvention may be encoded by nucleic acids located in separate vectors,or may be encoded by polynucleotides located in the same vector.

In an embodiment, a host cell is engineered to express a molecule of thepresent description. Host cells of the present description can beallogeneic or autologous with respect to a patient to be treated.

Yet another aspect of the invention relates to a pharmaceuticalcomposition comprising the dual specificity polypeptide moleculeaccording to the present invention, the nucleic acid(s) or theexpression vector(s) according to the present invention, or the cellaccording to the present invention, together with one or morepharmaceutically acceptable carriers or excipients. The compositions ofthe invention include bulk drug compositions useful in the manufactureof pharmaceutical compositions (e.g., impure or non-sterilecompositions) and pharmaceutical compositions (i.e., compositions thatare suitable for administration to a subject or patient) which can beused in the preparation of unit dosage forms. Such compositions comprisea prophylactically or therapeutically effective amount of theprophylactic and/or therapeutic dual specificity polypeptide molecule(agent) disclosed herein or a combination of the agent and apharmaceutically acceptable carrier. Preferably, compositions of theinvention comprise a prophylactically or therapeutically effectiveamount of one or more molecules of the invention and a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions preferably comprise the molecules eitherin the free form or as a salt. Preferably, the salts are pharmaceuticalacceptable salts of the molecules, such as, for example, the chloride oracetate (trifluoroacetate) salts. It has to be noted that the salts ofthe molecules according to the present invention differ substantiallyfrom the molecules in their state(s) in vivo, as the molecules are notsalts in vivo.

An embodiment of the present invention thus relates to a non-naturallyoccurring molecule according to the invention that has beensynthetically produced (e.g. synthesized) as a pharmaceuticallyacceptable salt. Methods to synthetically produce peptides and/orpolypeptides are well known in the art. The salts of the moleculesaccording to the present invention differ substantially from themolecules in their state(s) in vivo, as the molecules as generated invivo are no salts. Preferably, the salts are pharmaceutically acceptablesalts of the molecules. These salts according to the invention includealkaline and earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄)—Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂), CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂), such as, for example, the chloride or acetate(trifluoroacetate) salts.

In an aspect, a polypeptide described herein is in the form of apharmaceutically acceptable salt. In another aspect, a polypeptide inthe form of a pharmaceutical salt is in crystalline form.

In an aspect, a pharmaceutically acceptable salt described herein refersto salts which possess toxicity profiles within a range that isacceptable for pharmaceutical applications.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral—NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an aspect, pharmaceutically acceptable salts may increase thesolubility and/or stability of peptides of described herein. In anotheraspect, pharmaceutical salts described herein may be prepared byconventional means from the corresponding carrier peptide or complex byreacting, for example, the appropriate acid or base with peptides orcomplexes as described herein. In another aspect, the pharmaceuticallyacceptable salts are in crystalline form or semi-crystalline form. Inyet another aspect, pharmaceutically acceptable salts may include, forexample, those described in Handbook of Pharmaceutical Salts:Properties, Selection, and Use by P. H. Stahl and C. G. Wermuth(Wiley-VCH 2002) and L. D. Bighley, S. M. Berge, D. C. Monkhouse, in“Encyclopedia of Pharmaceutical Technology”. Eds. J. Swarbrick and J. C.Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995,pp. 453-499, each of these references is herein incorporated byreference in its entirety.

The invention also encompasses pharmaceutical compositions comprising adual specificity polypeptide molecule of the invention and a therapeuticantibody (e.g., tumor specific monoclonal antibody) that is specific fora particular cancer antigen, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,sodium phosphate, sodium acetate, L-Histidine, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. These compositions can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Generally, theingredients of compositions of the invention are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachet indicating the quantity of activeagent. Where the composition is to be administered by infusion, it canbe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the composition is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

Another aspect of the present invention then relates to the dualspecificity polypeptide molecule according to the invention, the nucleicacid or the expression vector according to the invention, the cellaccording to the invention, or the pharmaceutical composition accordingto the invention, for use in medicine. In general, the use of the dualspecificity polypeptide molecule depends on the medical context of thepeptide-antigen(s) that is/are recognized by said molecule, as is alsodescribed further below.

Preferred is the dual specificity polypeptide molecule according to theinvention, the nucleic acid or the expression vector according to theinvention, or the cell according to the invention, or the pharmaceuticalcomposition according to the invention, for use in the treatment orprevention of a disease or disorder selected from immunologicaldisorders, infectious disease, intoxication and cancers, includingtreatment or prevention of a variety of cancers or other abnormalproliferative diseases, including (but not limited to) the following:carcinoma, including that of the bladder, breast, colon, kidney, liver,lung, ovary, pancreas, stomach, prostate, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burketts'lymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, schwannomas, and osteosarcoma; andother tumors, including melanoma, xeroderma pigmentosum,keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma. Additional cancers may include but not be limited tofollicular lymphomas, carcinomas with p53 mutations, hormone dependenttumors of the breast, prostate and ovary, and precancerous lesions suchas familial adenomatous polyposis, and myelodysplastic syndromes. Inspecific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias), or hyperproliferative disorders, aretreated or prevented by the methods and compositions of the invention inthe ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. Inother specific embodiments, sarcoma, melanoma, or leukemia is treated orprevented by the methods and compositions of the invention.

The invention further relates to methods of eliciting an immune responsein a patient or subject comprising administering a therapeuticallyeffective amount of the dual specificity polypeptide molecule accordingto the invention or the pharmaceutical composition according to theinvention. In an aspect, a population of the dual specificitypolypeptide molecule according to the invention or the pharmaceuticalcomposition according to the invention is administered to a patient orsubject in need thereof.

The invention further relates to a method of killing target cells in apatient or subject comprising administering to the patient an effectiveamount of the dual specificity polypeptide molecule according to thepresent invention.

The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an inflammatory disorderin a subject further comprising, administering to said subject atherapeutically or prophylactically effective amount of one or moreanti-inflammatory agents according to the invention. The invention alsoprovides methods for preventing, treating, or managing one or moresymptoms associated with an autoimmune disease further comprising,administering to said subject a therapeutically or prophylacticallyeffective amount of one or more immunomodulatory agents according to theinvention. Infectious diseases that can be treated or prevented by themolecules of the invention are caused by infectious agents including butnot limited to viruses, bacteria, fungi, protozoae, and viruses. Viraldiseases that can be treated or prevented using the molecules of theinvention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, hantavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral meningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, Neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertussis,cholera, plague, diphtheria, chlamydia, S. aureus and legionella.

Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosome or malaria. Parasitic diseases thatcan be treated or prevented using the molecules of the invention inconjunction with the methods of the present invention, that are causedby parasites include, but are not limited to, chlamydia and rickettsia.

Examples of infectious agents and diseases include but are not limitedto bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Proteusvulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa), apathogen (e.g., B-lymphotropic papovavirus (LPV); Bordetella pertussis;Borne Disease virus (BDV); Bovine coronavirus; Choriomeningitis virus;Dengue virus; a virus, E. coli; Ebola; Echovirus 1; Echovirus-11 (EV);Endotoxin (LPS); Enteric bacteria; Enteric Orphan virus; EnterovirusesFeline leukemia virus; Foot and mouth disease virus; Gibbon ape leukemiavirus (GALV); Gram-negative bacteria; Helicobacter pylorii; Hepatitis Bvirus (HBV); Herpes Simplex Virus; HIV-I, Human cytomegalovirus; Humancoronovirus; Influenza A, B and C; Legionella; Leishmania mexicana;Listeria monocytogenes; Measles virus; Meningococcus; Morbilliviruses;Mouse hepatitis virus; Murine leukemia virus; Murine gamma herpes virus;Murine retrovirus; Murine coronavirus mouse hepatitis virus;Mycobacterium avium-M; Neisseria gonorrhoeae; Newcastle disease virus;Parvovirus B 19; Plasmodium falciparum; Pox Virus; Pseudomonas;Rotavirus; Salmonella typhiurium; Shigella; Streptococci; T-celllymphotropic virus 1; Vaccinia virus).

Yet another aspect of the present invention then relates to a method forthe treatment of a disease or disorder comprising administering atherapeutically effective amount of the dual specificity polypeptidemolecule according to the invention, the nucleic acid or the expressionvector according to the invention, the cell according to the invention,or the pharmaceutical composition according to the invention.

The dual specificity polypeptide molecule of the invention may be usedin a method of preventing or treating a disease or condition which isameliorated by administration of the dual specificity polypeptidemolecule. Such treatments may be provided in a pharmaceuticalcomposition together with one or more pharmaceutically acceptablecarriers or excipients. Therapeutic dual specificity polypeptidemolecules will usually be supplied as part of a sterile, pharmaceuticalcomposition which will normally include a pharmaceutically acceptablecarrier. This pharmaceutical composition may be in any suitable form,(depending upon the desired method of administering it to a patient). Itmay be provided in unit dosage form, will generally be provided in asealed container and may be provided as part of a kit. Such a kit wouldnormally (although not necessarily) include instructions for use. It mayinclude a plurality of said unit dosage forms. The pharmaceuticalcomposition may be adapted for administration by any appropriate route,such as a parenteral (including subcutaneous, intramuscular, orintravenous) route. Such compositions may be prepared by any methodknown in the art of pharmacy, for example by mixing the activeingredient with the carrier(s) or excipient(s) under sterile conditions.

In an aspect, peptides or other molecules described herein may becombined with an aqueous carrier. In an aspect, the aqueous carrier isselected from ion exchangers, alumina, aluminum stearate, magnesiumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g.,microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropyl methylcellulosePhthalate), starch, lactose monohydrate, mannitol, trehalose sodiumlauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, polymethacrylate, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

In an aspect, the aqueous carrier contains multiple components, such aswater together with a non-water carrier component, such as thosecomponents described herein. In another aspect, the aqueous carrier iscapable of imparting improved properties when combined with a peptide orother molecule described herein, for example, improved solubility,efficacy, and/or improved immunotherapy. In addition, the compositioncan contain excipients, such as buffers, binding agents, blastingagents, diluents, flavors, lubricants, etc. A “pharmaceuticallyacceptable diluent,” for example, may include solvents, bulking agents,stabilizing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likewhich are physiologically compatible. Examples of pharmaceuticallyacceptable diluents include one or more of saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, and the like as well ascombinations thereof. In many cases it will be preferable to include oneor more isotonic agents, for example, sugars such as trehalose andsucrose, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Pharmaceutically acceptable substances such as wettingor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, are also within the scope of thepresent invention. In addition, the composition can contain excipients,such as buffers, binding agents, blasting agents, diluents, flavors, andlubricants.

Dosages of the dual specificity polypeptide molecules of the presentinvention can vary between wide limits, depending upon the disease ordisorder to be treated, the age and condition of the individual to betreated, etc.; for example, a suitable dose range for a dual specificitypolypeptide molecule may be between 25 ng/kg and 50 μg/kg. A physicianwill ultimately determine appropriate dosages to be used.

Pharmaceutical compositions, vectors, nucleic acids and cells of theinvention may be provided in substantially pure form, for example atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% pure.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law. Althoughthe present invention and its advantages have been described in detail,it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims. The presentinvention will be further illustrated in the following Examples whichare given for illustration purposes only and are not intended to limitthe invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic overview over a preferred embodiment of thepresent invention, the human IgG1 Fc-containing dual specificitypolypeptide molecule. VD1, VD2=variable domains derived from antibody;VR1, VR2=variable domains derived from TCR, Link1, Link2=connectinglinkers; Cys-Cys=cysteine bridges.

FIG. 2 shows a schematic overview over 4 different constructs of IgGFc-containing dual specificity polypeptide molecules as tested in thecontext of the present invention. black=TCR-derived variable domains;light gray=antibody-derived variable domains; white=constant domainsderived from human IgG. Knob-hole mutations are indicated by a cylinder.Diabody molecules IA-ID are according to the invention.

FIG. 3 shows the HPLC-SEC analysis of different bispecific TCR/mAbmolecules with a molecular design according to the constructs depictedin FIG. 2, which were purified by a 2-column purification process. Themonomer contents of the different molecules were determined as follows.II: 93.84%; III: 96.54%; IV: 98.49%; IA_1: 95.48%; IA_3: 98.45%; ID_1:95.75%; IC_4: 95.22%; IC_5: 92.76%; ID_4: 99.31%; ID_5: 99.44%.

FIG. 4 shows the results of the potency assay with different bispecificTCR/mAb constructs (as shown in FIG. 2) designed as IgG4-basedmolecules. Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptideSLYNTVATL (SEQ ID No. 7) loaded T2 cells in the presence of increasingconcentrations of bispecific TCR (bssTCR) molecules. The bispecificTCR/mAb diabody molecule IA-IgG4 exhibited a higher potency than twoalternative dual specificity TCR/mAb molecules.

FIG. 5 shows the results of the potency assay with different bispecificTCR/mAb constructs (as shown in FIG. 2) designed as IgG1-basedmolecules. Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptideSLYNTVATL (SEQ ID No. 7) loaded T2 cells in the presence of increasingconcentrations of bispecific TCR (bssTCR) molecules. The bispecificTCR/mAb diabody molecules ID_1, IA_3 and IA1 exhibited markedly higherpotency than three alternative dual specificity TCR/mAb molecules.

FIG. 6 shows the results of the potency assay conducted with differentIgG1-based bispecific TCR/mAb constructs (as shown in FIG. 2) utilizingdifferent variable antibody domains both targeting the TCR-CD3 complex.Construct ID_1 comprises variable domains of the UCHT1(V9) antibodytargeting CD3, whereas the constructs ID_4 and ID_5 comprise variabledomains of the alpha/beta TCR-specific antibody BMA031.Jurkat_NFATRE_luc2 cells were co-incubated with HIV-peptide SLYNTVATL(SEQ ID No. 7) loaded T2 cells in the presence of increasingconcentrations of bispecific TCR (bssTCR) molecules.

FIG. 7 shows a schematic overview over the possible orientations of theVD and VR domains in the molecules of the present invention. VH:antibody-derived VH-domain, VL: antibody-derived VL-domain; Vα:TCR-derived Valpha; Vβ: TCR-derived Vbeta.

FIG. 8 shows the results of HPLC-SEC analysis of aggregates (HMWS—highmolecular weight species) within different bispecific TCR/mAb moleculesbased on IgG1. Aggregates were analyzed after purification and afterstorage of the molecules at 40° C. for 1 weeks and 2 weeks,respectively.

FIG. 9 shows the results of the potency assay conducted with differentbispecific TCR/mAb molecules based on IgG1. Potency was analyzed afterpurification and after storage of the molecules at 40° C. for 1 week and2 weeks, respectively. Stress storage at 40° C. did not lead tosignificant loss of potency of the molecules but a drastic increase inunspecific (i.e. target-independent) activation of Jurkat T cells wasdetected for the molecules III and IV.

FIG. 10 shows the results of a LDH-release assay with differentbispecific TCR/mAb constructs (as shown in FIG. 2) designed asIgG1-based molecules. PBMC isolated from a healthy donor wereco-incubated with HIV-peptide SLYNTVATL (SEQ ID No. 7) loaded T2 cellsin the presence of increasing concentrations of bispecific TCR (bssTCR)molecules. The bispecific TCR/mAb diabody molecules IA_3 and ID_1induced markedly higher lysis of target cells than three alternativedual specificity TCR/mAb molecules. As shown on the right hand sidedgraph none of the tested bispecific TCR/mAb constructs induceddetectable lysis of T2 cells loaded with irrelevant peptide (SEQ ID No.49).

FIG. 11 shows the results of a LDH-release assay with the bispecificTCR/mAb diabody construct IA_5 targeting tumor-associated peptidePRAME-004 (SEQ ID No. 49) presented on HLA-A*02. CD8-positive T cellsisolated from a healthy donor were co-incubated with cancer cell linesUACC-257, SW982 and U2OS presenting differing amounts ofPRAME-004:HLA-A*02-1 complexes on the cell surface (approx. 1100,approx. 770 and approx. 240 copies per cell, respectively, as determinedby M/S analysis) at an effector:target ratio of 5:1 in the presence ofincreasing concentrations of TCR/mAb diabody molecules. After 48 hoursof co-culture target cell lysis was quantified utilizing LDH-releaseassays according to the manufacturer's instructions (Promega).

FIG. 12 shows the results of a LDH-release assay with the bispecificTCR/mAb diabody constructs IA_5 and IA_6 utilizing a stability/affinitymaturated TCR and an enhanced version thereof, respectively, against thetumor-associated peptide PRAME-004 (SEQ ID No. 49) presented onHLA-A*02. CD8-positive T cells isolated from a healthy donor wereco-incubated with the cancer cell line U2OS presenting approx. 240copies per cell of PRAME-004:HLA-A*02-1 complexes or non-loaded T2 cells(effector:target ratio of 5:1) in the presence of increasingconcentrations of TCR/mAb diabody molecules. After 48 hours of coculturetarget cell lysis was quantified utilizing LDH-release assays accordingto the manufacturer's instructions (Promega).

FIG. 13 shows the results of a heat-stress stability study of theTCR/mAb diabody constructs IA_5 and IA_6 utilizing a stability/affinitymaturated TCR and an enhanced version thereof, respectively, against thetumor-associated peptide PRAME-004 (SEQ ID No. 49) presented onHLA-A*02. For this, the proteins were formulated in PBS at aconcentration of 1 mg/mL and subsequently stored at 40° C. for twoweeks. Protein integrity and recovery was assessed utilizing HPLC-SEC.Thereby the amount of high-molecular weight species was determinedaccording to percentage of peak area eluting before the main peak.Recovery of monomeric protein was calculated by comparing main peakareas of unstressed and stressed samples.

EXAMPLES Example 1 Design of Fc-Containing Bispecific TCR/mAb Diabodiesand Control Molecules.

Fc-containing bispecific TCR/mAb diabodies and control molecules (asdepicted in FIG. 2) were designed to specifically bind to the humanTCR-CD3 complex and to the peptide:MHC complex comprising theHIV-derived peptide SLYNTVATL (SQ ID No. 7) bound to HLA-A2*01. Fortargeting TCR-CD3 complex, VH and VL domains derived from theCD3-specific, humanized antibody hUCHT1(V9) described by Zhu et al.(Identification of heavy chain residues in a humanized anti-CD3 antibodyimportant for efficient antigen binding and T cell activation. JImmunol, 1995, 155, 1903-1910) or VH and VL domains derived from thealpha/beta TCR-specific antibody BMA031 described in Shearman et al.(Construction, expression and characterization of humanized antibodiesdirected against the human alpha/beta T cell receptor. J Immunol, 1991,147, 4366-73) and employed in the humanized version variant 10 (datagenerated in-house) were used. For targeting peptide:MHC complex, Valphaand Vbeta domains of the previously described stability and affinitymaturated, human single chain T-cell receptor 868Z11 disclosed by Aggenet al. (Identification and engineering of human variable regions thatallow expression of stable single-chain T cell receptors. PEDS, 2011,24, 361-372) were utilized.

In case of Fc-containing bispecific TCR/mAb diabodies DNA-sequencescoding for various combinations of VH and VL (corresponding to VD1 andVD2, respectively) and Va and Vb (corresponding to VR1 and VR2,respectively), as well as coding for linkers Link1 and Link2 wereobtained by gene synthesis. Resulting DNA-sequences were cloned in frameinto expression vectors coding for hinge region, CH2 and CH3 domainderived from human IgG4 [Accession#: K01316] and IgG1 [Accession#:P01857], respectively and were further engineered. Engineered wasperformed to incorporate knob-into-hole mutations into CH3-domains withand without additional interchain disulfide bond stabilization; toremove an N-glycosylation site in CH2 (e.g. N297Q mutation); tointroduce Fc-silencing mutations; to introduce additional disulfide bondstabilization into VL and VH, respectively, according to the methodsdescribed by Reiter et al. (Stabilization of the Fv Fragments inRecombinant Immunotoxins by Disulfide Bonds Engineered into ConservedFramework Regions. Biochemistry, 1994, 33, 5451-5459). An overview ofproduced bispecific TCR/mAb diabodies, the variants as well as thecorresponding sequences are listed in Table 1.

TABLE 1 Overview of all generated and evaluated Fc-containing bispecificTCR/mAb diabodies: KiH: Knob-into-hole; K/O: Fc-silenced, KiH-ds:Knob-into-hole stabilized with artificial disulfide-bond to connectCH3:CH3′, ds-hUCHT1(V9): disulfide-bond stabilized hUCHT1(V9) variabledomains; Link1: Linker connecting VR1 and VD1. Molecule TCR mAb SEQ IDsmodifications IA-IgG4 868Z11 hUCHT1(V9) SEQ ID No. IgG4 (KiH)  8 SEQ IDNo.  9 IA_1 868Z11 hUCHT1(V9) SEQ ID No. IgG1 (K/O, 10 KiH) SEQ ID No.11 IA_2 868Z11 hUCHT1(V9) SEQ ID No. IgG1 (K/O, 12 KiH-ds) SEQ ID No. 13IA_3 868Z11 ds-hUCHT1(V9) SEQ ID No. IgG1 (K/O, 14 KiH-ds) SEQ ID No. 15ID_1 868Z11 ds-hUCHT1(V9) SEQ ID No. IgG1 (K/O, 16 KiH-ds) SEQ ID No. 17IC_4 868Z11 hBMA031(var10) SEQ ID No. IgG1 (K/O, 18 KiH-ds) SEQ ID No.19 IC_5 868Z11 hBMA031(var10) SEQ ID No. IgG1 (K/O, 20 KiH-ds) SEQ IDNo. extended 21 Link1 ID_4 868Z11 hBMA031(var10) SEQ ID No. IgG1 (K/O,22 KiH-ds) SEQ ID No. 23 ID_5 868Z11 hBMA031(var10) SEQ ID No. IgG1(K/O, 24 KiH-ds) SEQ ID No. extended 25 Link1 IA_5 R16P1C10IhUCHT1(Var17) SEQ ID No. IgG1 (K/O, 43 KiH-ds) SEQ ID No. 44 IA_6R16P1010I#6 hUCHT1(Var17) SEQ_ID No. IgG1 (K/O, 45 KiH-ds) SEQ ID No. 46

Various control molecules exhibiting the same specificities wereconstructed Table 2 utilizing said VH, VL, Valpha and Vbeta domains incombinations with IgG1- or IgG4-derived constant domains comprisingengineered features as described above.

TABLE 2 Overview of all generated and evaluated Fc-containing bispecificcontrol molecules: KiH: Knob-into-hole; K/O: Fc-silenced. Molecule TCRmAb SEQ IDs modifications III-IgG4 868Z11 hUCHT1(V9) SEQ ID No. IgG4(KiH) 38 SEQ ID No. 39 IV-IgG4 868Z11 hUCHT1(V9) SEQ ID No. IgG4 40 SEQID No. 41 II 868Z11 hUCHT1(V9) SEQ ID No. IgG1 (K/O, KiH) 33 SEQ ID No.34 HI 868Z11 hUCHT1(V9) SEQ ID No. IgG1 (K/O, KiH) 35 SEQ ID No. 36 IV868Z11 hUCHT1(V9) SEQ ID No. IgG1 (K/O) 37 SEQ ID No. 42

Example 2 Production and Purification of Fc-Containing BispecificTCR/mAb Diabodies

Vectors for the expression of recombinant proteins were designed asmono-cistronic, controlled by HCMV-derived promoter elements,pUC19-derivatives. Plasmid DNA was amplified in E. coli according tostandard culture methods and subsequently purified usingcommercial-available kits (Macherey & Nagel). Purified plasmid DNA wasused for transient transfection of CHO-S cells according to instructionsof the manufacturer (ExpiCHO™ system; Thermo Fisher Scientific).Transfected CHO-cells were cultured for 6-14 days at 32° C. to 37° C.and received one to two feeds of ExpiCHO™ Feed solution.

Conditioned cell supernatant was harvested by centrifugation (4000×g; 30minutes) and cleared by filtration (0.22 μm). Bispecific molecules werepurified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equippedto perform affinity and size-exclusion chromatography in line. Affinitychromatography was performed on protein A columns (GE Lifesciences)following standard affinity chromatographic protocols. Size exclusionchromatography was performed directly after elution (pH 2.8) from theaffinity column to obtain highly pure monomeric protein using Superdex200 pg 16/600 columns (GE Lifesciences) following standard protocols.Protein concentrations were determined on a NanoDrop system (ThermoScientific) using calculated extinction coefficients according topredicted protein sequences. Concentration, if needed, and bufferexchange was performed using Vivaspin devices (Sartorius). Finally,purified molecules were stored in phosphate-buffered saline atconcentrations of about 1 mg/mL at temperatures of 2-8° C.

As therapeutic proteins shall exhibit reasonable stability upon acidicexposure to facilitate robust industrial purification processes thepercentage of monomeric protein eluting from the protein A capturecolumn was assessed (Table 3). It is obvious that the introduction ofstabilizing mutations into molecules as well as selection of distinctorientations of binding domains markedly impact the stability uponacidic exposure.

TABLE 3 Fraction of monomeric protein after acidic elution from capturecolumn: Monomer eluted from capture column Molecule (% of total peakarea) IA-IgG4 (VH-beta) n.d. IA_1 (VH-beta) 49 IA_2 (VH-beta) 54 IA_3(dsVH-beta) 63 ID_1 (alpha-dsVH) 46 IC_4 (VH-alpha) 62 IC_5 (VH-alpha)67 ID_4 (alpha-VH) 65 ID_5 (alpha-VH) 69 II 39 III 51 IV 76

After size exclusion chromatography, the purified bispecific moleculesdemonstrated high purity (>93% of monomeric protein) as determined byHPLC-SEC on MabPac SEC-1 columns (5 μm, 7.8×300 mm) running in 50 mMsodium-phosphate pH 6.8 containing 300 mM NaCl within an Agilent 1100system (see FIG. 3). Non-reducing and reducing SDS-PAGE confirmed thepurity and expected size of the different dual specificity TCR/mAbmolecules (data not shown).

Example 3 Specific and Target Cell-Dependent T Cell Activation Inducedby Fc-Containing TCR/mAb Diabodies

The potency of Fc-containing TCR/mAb diabodies with respect to T cellactivation was assessed using the T Cell Activation Bioassay (Promega).The assay consists of a genetically engineered Jurkat cell line thatexpresses a luciferase reporter driven by an NFAT-response element(NFAT-RE). Assays were performed according to the manufacturer. Briefly,T2 cells either loaded with the HIV-specific peptide SLYNTVATL (SEQ IDNo. 7) or left without peptide loading (unloaded control) weresubsequently co-cultured with Promega's modified Jurkat cells inpresence of increasing concentrations of bispecific TCR/mAb molecules.Jurkat reporter T cell activation was analyzed after 16-20 hours bymeasuring luminescence intensity.

Representative potency assay results are depicted for IgG4-based (FIG.4) and IgG1-based bispecific TCR/mAb molecules (FIG. 5), respectively.The data indicate that regardless of the IgG isotype of the constantdomains used, the Fc-containing TCR/mAb diabody constructs IA and IDshowed superior T cell activation compared to the alternative bispecificTCR/mAb constructs II, III and IV as measured by the magnitude ofactivation and/or respective EC50-values. Furthermore, the unspecific Tcell activation of Fc-containing TCR/mAb diabodies induced againstunloaded T2 cells was reduced or at least equal to the level ofunspecific activation observed for the alternative bispecific TCR/mAbconstructs. According to above results the dual specificity TCR/mAbdiabody molecules are preferred molecules for therapeutic interventionas they induce strong effector T cell activation in a highlytarget-dependent manner.

Furthermore LDH-release assay (Promega) was used to quantify thePBMC-mediated lysis of SLYNTVATL (SEQ ID No. 7) peptide-loaded T2 cellsinduced by the different bispecific TCR/mAb molecules (FIG. 10). In linewith the above results of the T Cell Activation Bioassay, again theFc-containing TCR/mAb diabody constructs IA and ID were superior overthe alternative bispecific TCR/mAb constructs II, III and IV asindicated by the increased absolute level of target cell lysis and thelower TCR bispecific concentration needed to achieve half-maximal (EC50)killing of target cells. As for TCR/mAb constructs II, III and IV, theTCR/mAb diabody constructs IA and ID did not induce lysis of T2 cellsloaded with irrelevant peptide (SEQ ID No. 49), proving thetarget-specific lysis to the T2 cells.

Example 4 Development of Fc-Containing Bispecific TCR/mAb Diabodies as aMolecular Platform

Fc-containing bispecific TCR/mAb diabody constructs were designed toserve as molecular platform to provide the scaffold for differentTCR-derived and mAb-derived variable domains targeting differentpeptide:MHC complexes and effector cell surface antigens, respectively.To validate the suitability as platform, the mAb-derived variabledomains were exchanged in a first set of molecules. The variable domainsof hUCHT1(V9) anti-CD3 antibody (construct ID_1) were replaced againstthe domains of the hBMA031(var10) anti-TCR antibody employing the samedomain orientation (constructs ID_4 and ID_5) or a different orientation(IC_4, IC_5) (see Table land FIG. 7 for details). Expression,purification and characterization of these molecules were performed asdescribed above. Purity and integrity of final preparations exceeded 92%according to HPLC-SEC analyses.

The potency assay results revealed target-dependent Jurkat reporter Tcell activation and minimal unspecific activity against unloaded T2cells for both antibody variable domains hUCHT1 (construct ID_1) andhBMA031 (constructs ID_4 and ID_5) supporting the platform suitabilityof the dual specificity TCR/mAb diabody constructs (FIG. 6). Notably,when the variable TCR and mAb domains of the constructs ID_4 and ID_5were switched on each polypeptide chain resulting in constructs IC_4 andIC_5 no T cell activation was observed (data not shown). The latterfinding indicate that despite bispecific TCR/mAb diabodies can be usedas platform construct for incorporating different TCR and mAb variabledomains a thorough optimization of the domain orientation is required toachieve optimal activity of the molecules.

Example 5 Stability of Fc-Containing Bispecific TCR/mAb Diabodies

Stability of the bispecific TCR/mAb molecules was initially assessedutilizing the Protein Thermal Shift Assay (Thermo Fisher Scientific)according to the instructions of the manufacturer using a 7500 Real timePCR system (Applied Biosciences). Briefly, purified molecules were mixedwith PTS buffer and PTS dye and subjected to a raising temperaturegradient constantly monitoring fluorescence of samples. Recordedfluorescence signals were analyzed using PTS software (Thermo FisherScientific) and melting temperatures (T_(M)) were calculated by thederivative method.

Stressed stability studies were conducted by storage of purifiedmolecules dissolved in PBS at 40° C. for up to two weeks. Samples wereanalyzed with regard to protein integrity using HPLC-SEC and potencyusing the T Cell Activation Assay (Promega) as described above.

As expected storage at 40° C. induced the formation ofaggregates/high-molecular weight species as determined by HPLC-SECanalyses (see FIG. 8). Results of potency assays of IgG1-based moleculesafter purification and incubation at 40° C. are shown in FIG. 9.Although neither of the tested molecules did show a significantreduction of potency after storage at 40° C., it was observed that thestressed molecules III and IV induced a significant amount of unspecific(i.e. target-independent) Jurkat T cell activation. In contrast, thebispecific TCR/mAb diabodies retained their target-dependent potency,despite the presence of some aggregates as seen in HPLC-SEC.

Example 6 Generation of Cancer-Targeting Bispecific TCR/mAb DiabodyMolecules

To further validate the platform capabilities of bispecific TCR/mAbdiabody constructs, the TCR-derived variable domains were exchanged withvariable domains of a TCR, which was stability/affinity maturated byyeast display according to a method described previously (Smith et al,2015, T Cell Receptor Engineering and Analysis Using the Yeast DisplayPlatform. Methods Mol Biol. 1319:95-141). The TCR variable domainsspecifically binding to HIV-derived peptide SLYNTVATL (SEQ ID No. 7) inthe context HLA-A*02 were exchanged with TCR variable domainsspecifically binding to the tumor-associated peptide PRAME-004 (SEQ IDNo. 49) bound to HLA-A*02. Furthermore, the variable domains of thehumanized T-cell recruiting antibody hUCHT1(V9) were exchanged againstvariable domains of hUCHT1(Var17), a newly humanized version of theUCHT1 antibody, resulting in the PRAME-004-targeting TCR/mAb diabodymolecule IA_5 (comprising SEQ ID No. 43 and SEQ ID No. 44). Expression,purification and characterization of this molecule was performed asdescribed in Example 2. Purity and integrity of final preparationexceeded 96% according to HPLC-SEC analysis.

Binding affinities of bispecific TCR/mAb diabody constructs towardsFRAME-004:HLA-A*02 were determined by biolayer interferometry.Measurements were done on an Octet RED384 system using settingsrecommended by the manufacturer. Briefly, purified bispecific TCR/mAbdiabody molecules were loaded onto biosensors (AHC) prior to analyzingserial dilutions of HLA-A*02/PRAME-004.

The activity of this PRAME-004-targeting TCR/mAb diabody construct withrespect to the induction of tumor cell lysis was evaluated by assessinghuman CD8-positive T cell-mediated lysis of the human cancer cell linesUACC-257, SW982 and U2OS presenting different copy numbers of PRAME-004peptide in the context of HLA-A*02 on the tumor cell surface(UACC-257—about 1100, SW982—about 770, U2OS—about 240 PRAME-004 copiesper cell, as determined by quantitative M/S analysis) as determined byLDH-release assay.

As depicted in FIG. 11, the PRAME-004-targeting TCR/mAb diabodyconstruct IA_5 induced a concentration-dependent lysis of PRAME-004positive tumor cell lines. Even tumor cells U2OS expressing as little as240 PRAME-004 copy numbers per tumor cell were efficiently lysed by thisTCR/mAb diabody molecule. These results further demonstrate that TCR/mAbdiabody format is applicable as molecular platform allowing to introducevariable domains of different TCRs as well as variable domains ofdifferent T cell recruiting antibodies.

Example 7 Engineerability of TCR/mAb Diabody Constructs

The variable TCR domains utilized in construct IA_5 were furtherenhanced regarding affinity towards PRAME-004 and TCR stability, andused for engineering into TCR/mAb diabody scaffold resulting inconstruct IA_6 (comprising SEQ ID No. 45 and SEQ ID No. 46). Expression,purification and characterization of TCR/mAb diabody molecules IA_5 andIA_6 were performed as described in example 2. Purity and integrity offinal preparations exceeded 97% according to HPLC-SEC analysis.

Potency of the stability and affinity enhanced TCR/mAb diabody variantIA_6 against PRAME-004 was assessed in cytotoxicity experiments with thetumor cell line U2OS presenting low amounts of PRAME-004:HLA-A*02 ornon-loaded T2 cells as target cells and human CD8-positive T cells aseffector cells.

As depicted in FIG. 12, the inventors observed and increased cytotoxicpotency of the TCR/Ab diabody molecule IA_6 comprising the variabledomains of the stability/affinity enhanced TCR variant when compared tothe precursor construct IA_5. For both constructs, IA_5 and IA_6, thePRAME-004-dependent lysis could be confirmed as no cytolysis oftarget-negative T2 cells was detected.

The protein construct were further subjected to heat-stress at 40° C.for up to two weeks to analyze stability of the PRAME-004-specificTCR/mAb diabody variants IA_5 and IA_6. HPLC-SEC analyses afterheat-stress revealed a significantly improved stability of the variantIA_6 when compared to the precursor construct IA_5 (see FIG. 13). Thetemperature-induced increase of high-molecular species (i.e. elutingbefore the main peak) of the constructs was less pronounced for IA_6than for IA_5. In line with this result, the recovery of intact,monomeric protein after heat-stress was 87% and 92% for IA_5 and IA_6,respectively.

These exemplary engineering data demonstrate that the highly potent andstable of TCR/mAB diabody constructs can further be improved byincorporating stability/affinity enhanced TCR variable domains resultingin therapeutic proteins with superior characteristics.

Example 8 Examples for Preferred Constructs

In addition to the HIV-specific TCR bispecific construct as describedherein (Seq ID No. 16 and Seq ID No. 17, in orientation D), theinvention further provides several other exemplary HIV-specificconstructs that were tested. These constructs are based on an improvedhumanized variants of the underlying antibody against CD3 (UCHT1) thatwere fused with the HIV-specific TCR 868 as disclosed herein in all fourpossible orientations (Seq ID No. 51 to Seq ID No. 58, in orientationsA-D).

The humanization of UCHT1 was performed using VH-1-46 and VK1-018 asacceptor frameworks for the heavy and light chain CDRs, respectively.J-segments selected were JK1 and JH4, for light and heavy chain,respectively.

The results as obtained are shown in the following Table 4:

V9 (Zhu et al, 1995) Present invention DRB1 score 1232 ~1190 Titre[mg/L] 0.75 3 Tm of F(ab) [° C.] 83.0 86.4 EC50 of effector cell 63 8activation [pM]

The data in table 4 shows that the inventive humanization is potentiallyless immunogenic (lower DRB1-score); the molecules are more stable(increase in melting temperature of about 3° C.); and more potent (˜8×decreased EC50), compared with the standard (V9) (for assay, see example3).

1. A method of eliciting an immune response in a patient who has cancer,comprising administering to a patient a dual specificity polypeptidemolecule comprising a first polypeptide chain and a second polypeptidechain, wherein: the first polypeptide chain comprises a first bindingregion of a variable domain (VD1) of an antibody specifically binding toa cell surface antigen of a human immune effector cell, and a firstbinding region of a variable domain (VR1) of a TCR specifically bindingto an MHC-associated peptide epitope, and a first linker (LINK1)connecting said domains; the second polypeptide chain comprises a secondbinding region of a variable domain (VR2) of a TCR specifically bindingto an MHC-associated peptide epitope, and a second binding region of avariable domain (VD2) of an antibody specifically binding to a cellsurface antigen of a human immune effector cell, and a second linker(LINK2) connecting said domains; wherein said first binding region (VD1)and said second binding region (VD2) associate to form a first bindingsite (VD1)(VD2) that binds a cell surface antigen of a human immuneeffector cell; said first binding region (VR1) and said second bindingregion (VR2) associate to form a second binding site (VR1)(VR2) thatbinds said MHC-associated peptide epitope; wherein said two polypeptidechains are fused to human IgG hinge domains and/or human IgG Fc domainsor dimerizing portions thereof; and wherein said two polypeptide chainsare connected by covalent and/or non-covalent bonds between said hingedomains and/or Fc-domains; and wherein said dual specificity polypeptidemolecule is capable of simultaneously binding the cell surface moleculeand the MHC-associated peptide epitope, wherein the order of the bindingregions in the two polypeptide chains is selected from VD1-VR1 andVR2-VD2 or VD1-VR2 and VR1-VD2, or VD2-VR1 and VR2-VD1 or VD2-VR2 andVR1-VD1, wherein the domains are either connected by LINK1 or LINK2; andwherein the cancer is selected from the group consisting of bladdercancer, breast cancer, colon cancer, kidney cancer, liver cancer, lungcancer, ovarian cancer, pancreatic cancer, stomach cancer, prostatecancer, cervical cancer, melanoma, thyroid cancer, brain cancer,leukemia, sarcoma, and lymphoma.
 2. The method according to claim 1,wherein the order of the binding regions in the polypeptide chains isselected from VD1-VR1 and VD2-VR2; and wherein the domains are connectedby LINK1 or LINK2, respectively.
 3. The method according to claim 1,wherein the linker-sequences LINK1 and/or LINK2 contain at least onesequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS, and TVLSSAS. 4.The method according to claim 1, wherein said first and secondpolypeptide chains further comprise at least a hinge domain and an Fcdomain or portions thereof derived from human IgG1, IgG2 or IgG4.
 5. Themethod according to claim 1, wherein said antibody-derived domains VD1and VD2 display an engineered disulfide bridge introducing a covalentbond between VD1 and VD2 and where said cysteines are introduced intoframework region (FR) 4 in case of VL and framework region 2 in case ofVH.
 6. The method according to claim 1, wherein said cell surfacemolecule induces the activation of immune cells, or is at least oneselected from the group consisting of CD3γ, CD3δ, CD3ε, CD4, CD7, CD8,CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b,CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61,CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163,CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI,TCRα/β, TCRγ/δ, and HLA-DR.
 7. A method of treating a patient withcancer, comprising administering to a patient a polypeptide moleculecomprising a first polypeptide chain and a second polypeptide chain,wherein the first polypeptide chain comprises a first hinge domainand/or a first Fc domain, wherein the first polypeptide chain comprisesat least 95% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 53, SEQ IDNO: 55, and SEQ ID NO: 57; wherein the second polypeptide chaincomprises a second hinge domain and/or a second Fc domain, wherein thesecond polypeptide chain comprises at least 95% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58; andwherein the cancer is selected from the group consisting of bladdercancer, breast cancer, colon cancer, kidney cancer, liver cancer, lungcancer, ovarian cancer, pancreatic cancer, stomach cancer, prostatecancer, cervical cancer, melanoma, thyroid cancer, brain cancer,leukemia, sarcoma, and lymphoma.
 8. The method according to claim 7,wherein said first polypeptide chain is fused to said second polypeptidechain by covalent and/or non-covalent bonds between the first hingedomain and the second hinge domain, and/or between the first Fc domainand the second Fc domain.
 9. The method according to claim 7, whereinsaid first and second Fc domains each comprise at least one effectorfunction silencing mutation.
 10. The method according to claim 7,wherein said first and second Fc domains each comprise a CH3 domaincomprising at least one mutation that facilitates the formation ofheterodimers.
 11. The method according to claim 7, wherein said firstand second Fc domains each comprise CH2 and CH3 domains comprising atleast two additional cysteine residues.
 12. The method according toclaim 7, wherein said cell surface molecule is at least one selectedfrom the group consisting of CD3γ, CD3δ, CD3ε, CD4, CD7, CD8, CD10,CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33,CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64,CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163,CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI,TCRα/β, TCRγ/δ, and HLA-DR.
 13. The method according to claim 7, whereinthe first polypeptide chain comprises at least 99% amino acid sequenceidentity to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, and SEQ ID NO:57; and wherein the second polypeptide chain comprises at least 99%amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 54, SEQ IDNO: 56, and SEQ ID NO:
 58. 14. A method of eliciting an immune responsein a patient who has cancer, comprising administering to a patient apolypeptide molecule comprising a first polypeptide chain and a secondpolypeptide chain, wherein the first polypeptide chain comprises a firsthinge domain and/or a first Fc domain, wherein the first polypeptidechain comprises at least 95% identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 55, and SEQ ID NO: 57; wherein the second polypeptidechain comprises a second hinge domain and/or a second Fc domain, whereinthe second polypeptide chain comprises at least 95% identity to an aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58; andwherein the cancer is selected from the group consisting of bladdercancer, breast cancer, colon cancer, kidney cancer, liver cancer, lungcancer, ovarian cancer, pancreatic cancer, stomach cancer, prostatecancer, cervical cancer, melanoma, thyroid cancer, brain cancer,leukemia, sarcoma, and lymphoma.
 15. The method according to claim 14,wherein said first polypeptide chain is fused to said second polypeptidechain by covalent and/or non-covalent bonds between the first hingedomain and the second hinge domain, and/or between the first Fc domainand the second Fc domain.
 16. The method according to claim 14, whereinsaid first and second Fc domains each comprise at least one effectorfunction silencing mutation.
 17. The method according to claim 14,wherein said first and second Fc domains each comprise a CH3 domaincomprising at least one mutation that facilitates the formation ofheterodimers.
 18. The method according to claim 14, wherein said firstand second Fc domains each comprise CH2 and CH3 domains comprising atleast two additional cysteine residues.
 19. The method according toclaim 14, wherein said cell surface molecule is at least one selectedfrom the group consisting of CD3γ, CD3δ, CD3ε, CD4, CD7, CD8, CD10,CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33,CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64,CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163,CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI,TCRα/β, TCRγ/δ, and HLA-DR.
 20. The method according to claim 14,wherein the first polypeptide chain comprises at least 99% amino acidsequence identity to a sequence selected from the group consisting ofSEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, andSEQ ID NO: 57; and wherein the second polypeptide chain comprises atleast 99% amino acid sequence identity to a sequence selected from thegroup consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 25, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, and SEQ ID NO: 58.